SUMMARY There is a continued need for therapies to regenerate muscle and/or prevent muscle loss. The current R21 proposal challenges the dogma that myonuclei are unable to replicate in order to replace lost nuclei or support hypertrophic growth. This high-risk, high-reward proposal tests the overall hypothesis that myonuclei are capa- ble of replication, thus answering a long-standing unresolved question. This hypothesis was formed by intri- guing evidence showing BrdU incorporation by myonuclei in satellite cell-depleted muscle as well as the ap- parent lifelong maintenance of myonuclei in the absence of satellite cells. Additional support for our overall hy- pothesis comes from pioneering studies demonstrating the capability of mammalian myocytes to de- differentiate and re-enter the cell cycle, and the discovery that other cell types once thought to be post-mitotic have the ability to replicate. To test the overall hypothesis, the specific aim is designed to test if myonuclei have the ability to replicate during regular cage activity or during overload-induced hypertrophy. The proposed approach is technically feasible because of the assembled expertise of the investigative team. The approach uses a novel transgenic mouse that allows for GFP-labeling of myonuclei specifically during a defined period of time such that no new GFP labeling will occur during the proposed interventions. During the intervention mice will be administered deuterium oxide (D2O) via drinking water, which labels any newly synthesized DNA during a period of time when new myonuclei from other cellular sources will not contain GFP. Following the interven- tion, GFP-labeled myonuclei will be isolated by FACS, and D2O incorporation determined by mass spectrome- try in GFP+ cells. Given the high specificity of GFP labeling with this design, this innovative approach allows for unambiguously determining if any myofiber nuclei replicated and under what condition(s). The project is highly significant because evidence supporting the hypothesis would radically transform the field's current under- standing of the basic biology of skeletal muscle. Such evidence would make myonuclei a novel therapeutic tar- get to prevent muscle loss or increase muscle growth. The project is innovative because it combines a novel myofiber-specific Tet-ON mouse and D2O labeling to unambiguously assess myonuclear DNA synthesis. If successful, the proposed research would reverse a long-standing dogma and create new areas of investigation and clinical development. Future studies would characterize additional parameters of myoncuclei turnover, as well as mechanistic studies to determine how or when myonuclei replicate. The resulting impact is a new ave- nue for the development of innovative treatments to combat muscle loss with age and diseases of muscle wasting.